Phosphatidylserine in addition to phosphatidylethanolamine is an in vitro target of the mammalian Atg8 modifiers, LC3, GABARAP, and GATE-16
Phosphatidylserine in addition to phosphatidylethanolamine is an in vitro target of the mammalian Atg8 modifiers, LC3, GABARAP, and GATE-16
复制标题
DOI：
10.1074/jbc.m505888200
复制
发表时间：
2006-02-10
影响因子：
4.8
通讯作者：
Kominami, E
中科院分区：
文献类型：
--
作者：
Sou, Y;Tanida, I;Kominami, E
在酵母中，磷脂酰乙醇胺是自噬所必需的泛素化样反应中Atg 8修饰剂的靶标。三种人类Atg 8（hAtg 8）同源物，LC 3，GABARAP和GATE-16，已被表征为在由hAtg 7（E1样酶）和hAtg 3（E2样酶）介导的反应中的修饰剂，如在酵母Atg 8脂化中，但它们的最终靶点尚未确定。最近的一项研究将COS 7细胞与[ C-14]乙醇胺孵育48小时，结果表明磷脂酰乙醇胺是LC 3的靶点。然而，由于孵育时间较长，这些结果并非结论性的。为了鉴定Atg 8同源物的磷脂靶点，我们使用纯化的重组Atg蛋白和脂质体在体外重建哺乳动物Atg 8同源物的缀合系统。每个纯化的突变体Atg 8同系物与暴露的C-末端Gly形成E1-底物中间体与hAtg 7通过硫酯键在ATP依赖性的方式和形成E2-底物中间体与hAtg 3通过硫酯键依赖于ATP和hAtg 7。在存在hAtg 7、hAtg 3、ATP和脂质体的情况下观察到每种Atg 8同系物的缀合形式。除了磷脂酰乙醇胺之外，使用合成磷脂脂质体的体外缀合实验表明，磷脂酰丝氨酸也是LC 3、GABARAP和GATE-16的靶点。相反，从内源性LC 3-磷脂缀合物中释放的hAtg 4 B消化的磷脂的薄层色谱显示，磷脂酰乙醇胺，而不是磷脂酰丝氨酸，是LC 3在体内的主要靶磷脂。体外和体内反应之间的差异表明，可能存在参与内源性LC 3缀合系统的选择性因子。
In yeast, phosphatidylethanolamine is a target of the Atg8 modifier in ubiquitylation-like reactions essential for autophagy. Three human Atg8 ( hAtg8) homologs, LC3, GABARAP, and GATE-16, have been characterized as modifiers in reactions mediated by hAtg7 ( an E1-like enzyme) and hAtg3 ( an E2-like enzyme) as in yeast Atg8 lipidation, but their final targets have not been identified. The results of a recent study in which COS7 cells were incubated with [ C-14] ethanolamine for 48 h suggested that phosphatidylethanolamine is a target of LC3. However, these results were not conclusive because of the long incubation time. To identify the phospholipid targets of Atg8 homologs, we reconstituted conjugation systems for mammalian Atg8 homologs in vitro using purified recombinant Atg proteins and liposomes. Each purified mutant Atg8 homolog with an exposed C-terminal Gly formed an E1-substrate intermediate with hAtg7 via a thioester bond in an ATP-dependent manner and formed an E2-substrate intermediate with hAtg3 via a thioester bond dependent on ATP and hAtg7. A conjugated form of each Atg8 homolog was observed in the presence of hAtg7, hAtg3, ATP, and liposomes. In addition to phosphatidylethanolamine, in vitro conjugation experiments using synthetic phospholipid liposomes showed that phosphatidylserine is also a target of LC3, GABARAP, and GATE-16. In contrast, thin layer chromatography of phospholipids released on hAtg4B-digestion from endogenous LC3-phospholipid conjugate revealed that phosphatidylethanolamine, but not phosphatidylserine, is the predominant target phospholipid of LC3 in vivo. The discrepancy between in vitro and in vivo reactions suggested that there may be selective factor( s) involved in the endogenous LC3 conjugation system.